Stabilized microspheres and methods of preparation

ABSTRACT

Stabilized microspherical particles having hydrophobic liquid cores prepared as oil-in-water microemulsions. The particles are stabilized by a surface layer comprising an amphiphilic compound and may be functionalized to allow covalent coupling of a ligand to the surface of the particle. When used as tracers in assays, a water insoluble dye may be incorporated in the core liquid of the microparticles.

This is a division of application Ser. No. 08/343,305, filed Nov. 22,1994, now U.S. Pat. No. 5,580,735 which is a division of applicationSer. No. 08/001,907, filed on Jan. 4, 1993, now U.S. Pat. No. 5,393,527.

FIELD OF THE INVENTION

The present invention relates to oil-in-water emulsions, methods formaking such emulsions and methods for using the microspherical particlesin diagnostic assays.

BACKGROUND OF THE INVENTION

Emulsions are fluid systems comprising two immiscible liquid phases withone liquid dispersed as droplets in the other. The dispersed liquid isreferred to as the dispersed phase and the liquid in which it isdispersed is referred to as the continuous phase. Microemulsions areemulsions in which the dispersed phase droplets are very small,typically about 0.01 μm to about 0.2 μm. The choice of liquids for thetwo phases and the surfactants used determine whether the microemulsionis oil-in-water (o/w), water-in-oil (w/o) or anhydrous. Microemulsionsare generally more stable than emulsions with discontinuous phasescomprising larger droplets because the interfacial tension between theoil and water phases is significantly lower. That is, in microemulsionsthere is a reduced tendency for the droplets of the dispersed phase tocoalesce.

Emulsions are generally made by subjecting the component liquids,including an emulsifier, to high shear forces. The forces may bemechanical, such as vigorous stirring or forcing the mixture through asmall orifice. Alternatively, ultrasonic emulsification may be used toeffect cavitation in the liquids with high local shear. Asmicroemulsions provide a means for maintaining, in stable aqueoussolution, substances which are otherwise insoluble in an aqueous phase,they are of interest as potential drug delivery systems for lipophilicdrugs. Surface modifications on the dispersed phase droplets have beenfound to be useful for altering the physicochemical and biochemicalproperties of the colloidal droplets, including the kinetics of bloodclearance and tissue distribution. (K. Iwamoto et al. 1991. J.Pharmaceutical Sciences 80, 219.)

As used herein, the term "water" in reference to emulsions means a polarhydrophilic liquid and is not limited to water per se. Similarly, theterm "oil" in reference to emulsions means any nonpolar hydrophobicliquid. The terms "microspherical emulsion", "microemulsion" and relatedterms refer to stable emulsions in which the droplets of the dispersedphase are very small. "Microparticle", "microsphere", "particle","microspherical particle" and related terms refer to the droplets of thedispersed phase of the microemulsion, which may or may not be emulsifiedin an aqueous phase. "Functionalized" particles, microparticles,droplets or microspheres have at least one amphiphilic component in thesurface layer which includes a reactive group suitable for covalentlycoupling the microparticle to a ligand.

A ligand specifically recognizes and binds to a receptor molecule. Theligand and its receptor are referred to as a specific binding pair. Thespecificity of binding of a ligand and receptor can be used in assaysfor detection of an analyte which is either a ligand or receptor.Ligand/receptor pairs include, as examples, antigens or haptens andantibodies, complementary nucleic acids, biotin and avidin/streptavidin,and carbohydrates and lectins.

Methods for production of the microspheres of the invention are similarto the ethanol injection methods previously known for the preparation ofliposomes. However, such methods have not heretofore been adapted forthe production of particles with hydrophobic liquid cores andamphiphilic monolayers on the surface. The ethanol injection method forproduction of single bilayer liposomes encapsulating an aqueous mediumis described by S. Batzri and E. Korn (1973. Biochim. Biophys. Acta 298,1015.) and in Liposomes--A Practical Approach (IRL Press, R.R.C. New,ed., pg. 63). U.S. Pat. No. 5,100,591 describes methods for productionof liposomes which incorporate an amphoteric, water-insoluble substancesuch as amphotericin B into the membrane with the phospholipids.

The stabilized microparticles of the invention are useful in a widevariety of applications where a hydrophobic particle core is desirable.Liposomes have previously been used for many of these applications, withvarious problems and disadvantages. In the present invention, the liquidhydrophobic core allow a efficient incorporation of water-insolublecompounds for use in cosmetics (e.g., dyes and fragrances), foods (e.g.,oils and flavors) and agriculture (e.g., insecticides and herbicides).Water-insoluble drugs may also be included in the core of themicroparticles to improve drug delivery and stability in therapeuticapplications. Such drug-containing microspheres are capable ofincorporating more of the drug than the "pharmacosomes" of the priorart, which can only encapsulate the water-insoluble drug in therelatively small hydrophobic regions within the membrane bilayer ratherthan in the relatively large aqueous core. See Perrett, et al. 1991. J.Pharm. Pharmacol. 43, 154; Hamann, et al. 1989. 5th Internat. Conf.Pharm. Tech, 1, 99; Hamann, et al. 1987. Acta Pharm. Technol. 33, 67 fordiscussions regarding incorporation of drugs into liposomes. Further,amphiphilic drugs such as amphotericin B or insecticidal/herbicidalfatty acids may be incorporated as amphiphiles in the surface monolayerof the inventive microspheres for therapeutic and agriculturalapplications.

As the surface charge of the present stabilized microspheres can beincreased by adjusting the types and amounts of amphiphilic drugs oramphiphilic lipids in the surface monolayer, they can be made to movewith an electric potential while carrying pharmaceutical agents. Theymay therefore also be useful for delivery of drugs by iontophoresis.

SUMMARY OF THE INVENTION

Oil-in-water emulsions containing stabilized microspherical particles asthe dispersed phase are provided. The microparticles comprise a liquidhydrophobic core with a surface coating of an amphiphilic compound. Themicroparticles or droplets of the dispersed phase may optionally befunctionalized by inclusion of a reactive group on the surface forcoupling to antibody, antigen, hapten or oligonucleotide ligands.Ligand-derivatized microparticles are useful as reagents in diagnosticassays involving binding of a ligand to its receptor and can replaceother types of particulate reagents typically used in diagnostic assays,e.g., latex particles, metal sol particles, magnetic beads, colloidalparticles or liposomes. If a water-insoluble dye is included in the coreliquid, the microemulsions may also be used as tracers in the diagnosticassays to provide a reporter or detector function. The microsphericalparticles of the invention have the advantage of incorporating a higherconcentration of dye than is possible using liposomes, as liposomesencapsulate a .large amount of aqueous medium in addition to theentrapped dye. They are also more intensely colored than colored latexparticles, thus improving assay sensitivity.

The microspherical particles of the oil-in-water emulsions havestabilized, non-drying, liquid cores and will readily rehydrate toapproximately their original size upon reconstitution from a driedstate. This is an improvement when compared to many other particle typessuch as latex, which do not resuspend easily after drying. In addition,the microparticles remain stable when stored at room temperature. Whenused as tracers, the microspheres will not leak the water-insoluble dyecontained in the core into the surrounding aqueous medium. In contrast,liposomes containing water-soluble dyes tend to hydrate slowly and leaksubstantial amounts of dye upon reconstitution from a dried state. Thisdifference in rehydration time is most likely due to the fact that thedroplets need only be hydrated at their surfaces to function in anaqueous environment, whereas liposomes must be hydrated on their lipidbilayer surfaces as well as in their interior to function in an aqueousenvironment after lyophilization. Rapid rehydration with minimal loss ofsignal are highly desirable characteristics for tracer reagents,particularly when they are used in immunocapillary orimmunochromatographic diagnostic tests.

DETAILED DESCRIPTION OF THE INVENTION

The stabilized microspherical particles of the invention are prepared asoil-in-water (o/w) microemulsions using water-insoluble compounds andmixtures of amphiphiles. The amphiphiles coat the dispersed droplets ofthe water-insoluble compound and stabilize them in the aqueous phase.Dyes soluble in the water-insoluble compound may optionally be included.In general, the microemulsions may be prepared by any of the methodsknown in the art, but certain modifications are preferred. Because theamphiphiles are not soluble in the water-insoluble compound, a cosolventis preferably initially included during the preparation of theemulsions. The cosolvent is then removed at a later stage of thepreparation process.

The water-insoluble compounds which form the cores of the microsphericalparticles are liquid at room temperature and preferably have a highcapacity to dissolve water-insoluble dyes if the microemulsions are tobe used as tracers. Preferably, they withstand vaporization underconditions used for lyophilizing aqueous solutions and have a densityequal to or slightly higher than the density of the aqueous environmentin which they will be emulsified, i.e., usually about 1.0 or slightlyhigher. Water-insoluble compounds having these properties, whenstabilized in a microemulsion, behave like solid particles when themicroemulsion is lyophilized, The preferred water-insoluble compound foruse in preparing the inventive microemulsions is polydimethyldiphenylsiloxane, such as GE Silicone SF 1154 available from General ElectricCo., Waterford, N.Y. This silicone fluid has a density of 1.05, lowviscosity and is non-volatile. Other silicones may also be used in theinvention, for example GE SF 1265, or Huls America (Piscataway, N.J.)fluorosilicones PS 181 and PS 182. When fluorosilicones are used, a 1:1mixture of DMF (dimethylformamide) and ethanol is required to bring theother components into solution.

The continuous phase of the microemulsions may be any aqueous compoundsuitable for the desired application. Examples of aqueous continuousphase compounds include water and various buffers as are known in theart.

The amphiphiles used to prepare the microemulsions stabilize themicroparticles to the aqueous environment by preventing coalescence ofthe oil droplets, forming a layer on the surface of the dispersed oildroplet. This layer is believed to be a monolayer, in contrast to thebilayer membranes formed in liposomes. This structure is supported byexperimental analysis showing that the ratio of maleimide on the surfaceof the microparticles to the total amount of maleimide in the particleis 1:1 (see the Examples). In contrast, liposomes give a ratio of 2:1 orhigher depending on the size of the liposome and the number of bilayersit contains. Preferably, a mixture of amphiphiles is used. Suitableamphiphiles include the lipids typically used in the preparation ofliposomes. Preferred amphiphiles are phospholipids such as phosphatidylcholine, phosphatidyl glycerol and phosphatidyl ethanolamine. Mostpreferred are the distearoyl phospholipids, e.g., distearoylphosphatidyl choline (DSPC), distearoyl phosphatidyl glycerol (DSPG) anddistearoyl phosphatidyl ethanolamine (DSPE). DSPE may be derivatized toinclude a maleimidyl caproate moiety (DSPE-MC) to functionalize themicroparticle for coupling to a ligand. The charge of the microparticlesmay be adjusted for a particular pharmaceutical or iontophoresisapplication by increasing or decreasing the amount of DSPG or DSPE, orby treating the maleimide with MESA.

When the microparticles are to be functionalized, it is preferred thatthe amphiphilic component be a mixture of amphiphiles which includes atleast one amphiphile which can be covalently linked to the selectedligand. The coupling amphiphile may be a derivatized phospholipid whichcarries a functional group suitable for covalent coupling of the liganddirectly to the surface of the microparticle, e.g., a maleimidylderivative of the phospholipid or thiocholesterol. Covalent methods aresuitable for coupling antigen, antibody, hapten and oligonucleotideligands to the microparticle, the oligonucleotides preferably beinglinked through thio groups to a maleimide function on the phospholipid.Maleimide functionalized phospholipids may also be coupled to avidin,streptavidin or biotin which are then noncovalently bound to a biotin oravidin/streptavidin-derivatized ligand, resulting in indirect couplingof the ligand to the microparticle. H. C. Loughrey, et al. 1990. J.Immunol. Mtds. 132, 25. Biotinylated protein or oligonucleotide ligandsmay also be indirectly coupled to the surface of the microparticle viaan anti-biotin antibody covalently coupled to the surface phospholipids.

Alternatively, an amphiphilic ligand may be included in the mixture ofstabilizing amphiphiles which comprises the coating on the surface ofthe droplet. For example, cardiolipin, a four-chain phospholipid, is asuitable amphiphile for preparation of the microemulsions and is alsouseful as an antigen ligand for syphilis serology testing.Microparticles which include cardiolipin on their surfaces can be usedin agglutination immunoassays for detection of anti-syphilis antibodies.

Cholesterol may be included with the amphiphiles but is generally notrequired as it is for liposomes because the amphiphiles form a monolayeron the microdroplet rather than a collection of bilayers as inliposomes. As the function of cholesterol in liposome membranes is tointercalate in and stabilize the liposome bilayers, it is optional inthe present invention. Preferably, cholesterol is not included when themicroparticles are used as tracers, as its presence may increasebackground staining and baseline levels of agglutination in certainsolid phase immunoassays. It has also been found that increased amountsof DSPG increase the surface charge of the particles and preventaggregation after antibody coupling, thereby reducing background andfalse positives when the microparticles contain a dye. Alternatively,treatment of the particles with MESA (2-mercaptoethanesulfonic acid)after coupling to the ligand also reduces background and falsepositives.

Water-insoluble dyes are optionally incorporated into the droplets ofthe o/w microemulsion to prepare tracer compositions for detectinganalytes in diagnostic assays. Detectable tracers and tracers comprisingdyes are well known in the art of specific binding assays, i.e., assayswhich involve binding of a ligand and its specific binding pair member.The water-insoluble compounds of the particle core are capable ofdissolving the water-insoluble dyes at high concentration, therebyimproving the sensitivity of the diagnostic assay. Suitablewater-insoluble dyes are those known in the art for incorporation intotracers, e.g., encapsulation into liposomes or incorporated into latexparticles. Different dyes may be incorporated into separatemicroparticle preparations which may then be mixed for use in assays formultiple analytes or where multisignal readouts are desired. A preferreddye for use in the invention is Sudan Black B.

When Sudan Black B (SBB) is incorporated into the microsphericalparticles, it is preferred that the dye first be purified. Purificationand isolation of the major components of SEB are described by U.Pfuller, et al. 1977. Histochemistry 54, 237 and U. Pfuller, et al.1977. Verb. Anat. Gas. 71, S 1439. Using preparative thin layerchromatography with chloroform as the mobile phase, the major fastmoving (i.e., more hydrophobic) component can be isolated. Thiscomponent is herein referred to as "Fast SBB." Fast SBB may also beisolated by chromatography on a silica column usingdichloromethane:hexane 80:20 as the solvent or from TLC plates run indichloromethane. Dichloromethane in general provides better separationof Fast SBB from Slow SBB than does chloroform. Sudan Black Bcommercially available from Eastman Kodak, Rochester, N.Y. is suppliedas 98% pure, but Fast SBB comprises about 17% of the total weight(assuming 100% yield on purification). The use of Fast SBB provides fora more stable particle, incorporation of higher amounts of dye, and amore stable maleimide function than is possible using unpurified SBB.Purified Fast SBB will not partition into the aqueous phase eitherduring particle formation or during rehydration of lyophilized particlesand is therefore preferred for stability and intensity of the signal.

The purity of the Fast SBB preparation may be approximated aftersolubilization in ethanol by the absorbance ratio at 700 and 600 nm(extinction coefficient 24000):

    ______________________________________                                                    A700/A600                                                         ______________________________________                                        Impure SBB    .23                                                             Slow SBB      .27                                                             Fast SBB      .08                                                             ______________________________________                                    

In one embodiment, silicone microemulsions are made by dropwise additionof an ethanol solution of lipids, silicone and dye (optional) into theaqueous phase while mixing on a vortex mixer. This method results in agradual increase in ethanol concentration from 0% to a finite level,usually less than about 50%. The ratio of ethanol to aqueous isapparently not critical within this range. Particles are preferablyproduced by mixing the ethanol solution with the aqueous phase toachieve a final concentration of ethanol of about 20-40%. Mostpreferably, the ethanol solution and the aqueous are mixed in a ratio ofabout 1:2, resulting in a final concentration of ethanol of about 30%.However, the ethanol concentration may be as high as about 50% if it isdried (e.g., using a 4A molecular sieve to remove IPA, benzene, water,etc.) and the lipid concentration is reduced. After formation of themicroparticles, the ethanol is preferably removed, e.g., byultrafiltration, column chromatography or dialysis. Removal of theethanol is not essential for coupling the microparticles to a ligand,but it is preferred. If the particles are to be coupled to a ligand,they are preferably concentrated prior to coupling. This method is rapidand simple for small-scale production of microspheres, but is notamenable to large scale production.

A large-scale method for production of the microspheres uses pumps orsyringes to bring the ethanol solution into contact with the aqueousphase, preferably with fluid streams coming together in a "Y"connection. This method is advantageous in that it can be automated. Forexample, pumps running at different speeds or a Zymark Corp. MASTERLABORATORY STATION with syringes of appropriate size controlled bystepper motors to deliver the components at a selected ratio provide asystem that is automated, scalable and allows control of the ratio ofethanol to aqueous. This ensures consistent particle formation andparticle size distribution. Such an automated system also allows forformation of the particles at a fixed ratio of ethanol solution toaqueous, which results in a fixed concentration of particles. Largervolumes of microparticles may be produced by running the pumps/syringesfor longer time periods, increasing the sizes of the syringes, etc. Nodeterioration was observed when 10 and 25 ml syringes were used in placeof 3 ml syringes. The syringe system is preferred because it may providethe additional benefit of increased shear force and mixing generated bythe expulsion of the two fluid streams through the narrow syringeopening.

In both preparation methods, particles of defined size distribution lessthan 1 μm and typically less than 250 nm are formed without extrusion orsonication, as would be required for formation of liposomes of definedsize. The ratio of lipids to silicone, the concentration of lipids andsilicone in the ethanol and the ratio of organic volume (ethanolsolution) to aqueous volume determine the particle size of themicroemulsion. These ratios are adjusted to obtain the desired particlesize during the formation of the microemulsion as, in contrast toliposomes, the microparticles cannot be resized after they have beenmade. Most preferably, the microemulsions have a particle size under 200nm.

The organic cosolvent is a water miscible organic solvent which performsthe function of solubilizing all of the components. Examples includeshort chain alcohols such as ethanol and dimethylformamide (DMF).Individually, the components are either insoluble in each other orinsoluble in the organic cosolvent. For example, 1) lipids are notsoluble in silicone and 2) DSPG and DSPE are not soluble in ethanol.However, as a combined system these components are soluble. Thewater-insoluble dye, if present, is soluble in silicone or lipids. Ithas also been found that the silicone allows incorporation of as much asten times the amount of dye which can be incorporated without silicone(mM dye/μM Pi without silicone=0.3, with silicone=3.0).

Preferably, when functionalized microparticles are to be coupled to aligand they are typically first concentrated to about 100 nM maleimideper ml to increase the efficiency of coupling. The particles may beconcentrated by dialysis, lyophilization and reconstitution or othersuitable means known in the art. If desired, microparticles may becoupled to the ligand using appropriate protocols as described above,including forming a covalent linkage to a maleimide function on anamphiphile in the particle coating. When a protein is to be coupled,thiols are reduced prior to coupling. This protein may be the liganditself (direct coupling) or it may be avidin, biotin or antibody for usein noncovalent coupling of the ligand to the microparticle.

In a preferred embodiment for producing a reagent for immunoassay, anantibody ligand is coupled to the particle at pH 8 and remaining freeantibody is removed by column chromatography. A SEPHAROSE FAST FLOWcolumn (Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.)equilibrated in 30 mM MOPSO (3- N-Morpholino!-2-hydroxy-propanesulfonicacid), 10 mM EDTA, 100 mM glucose, and 0.2% NAN₃, pH 6.8, is preferred.This buffer gives a good yield as a result of minimal sticking ofparticles to the column. Sucrose may be substituted for glucose in thebuffer without adversely affecting subsequent lyophilization andreconstitution of the particles. Additives such as glycerol/DMSO shouldbe avoided due to unsatisfactory performance in assays afterlyophilization and reconstitution. Dextran and beta hydroxy propylcyclodextrin additives are optional and do not adversely affectperformance of the particles after lyophilization.

The microparticles may be lyophilized for storage by freezing themicroemulsion and lyophilizing it. The microemulsion may either befrozen slowly (e.g., at about -40° C. in the lyophilizer) or morerapidly in liquid nitrogen (about -150° C.). In one embodiment, thefrozen microemulsions are lyophilized such that warming from about -40°C. to +25° C. occurs over a 4 to 12 hour period under vaccuum. The vialsare then held at +25° C. until the lyophilization is complete. Thelyophilized particles show improved performance in diagnostic assayswhen the vials are closed under vacuum in a low humidity environmentafter lyophilization. Conveniently, this can be done in the lyophilizer.This procedure reduces the background when the microemulsions are usedas tracers in specific binding assays, thereby improving assaysensitivity and providing a cleaner system for evaluating and optimizingassay parameters.

The particles are preferably lyophilized in the presence of at least 1%of a stabilizing protein to improve stability of the functionalizedmicrospherical particles during storage or treatment at elevatedtemperatures. Preferably the stabilizing protein is at least 1% BSA,more preferably 5% BSA (Pentex Fraction V BSA, Miles Laboratories,Kankakee, Ill.), but other proteins known in the are for this purposeare also suitable, e.g., casein. Particle concentration and the ratio ofparticles to stabilizing protein affect the level of background seen indiagnostic tests using reconstituted microemulsions. It has been notedthat too little or too much BSA (13% or more) increases the backgroundin the malaria test described in Example 3. The ratio of stabilizingprotein to particles (quantitated by the amount of P_(i)) is preferablygreater than 28 mg protein/l μM P_(i) and less than 200 mg/l μM P_(i).

The lyophilized microparticles, stored in closed containers, may berapidly reconstituted (i.e., resuspended) in any suitable aqueousmedium, for example water or a buffer. Suitable buffers include, forexample, MOPSO/glucose, MOPSO/glucose/BSA and borate buffers. Thereconstituted particles are similar in size to particles which have notbeen lyophilized. An appropriate dilution of particles for a desiredapplication can be obtained by adjusting the amount of aqueous mediumadded to reconstitute the particles. Samples in vials which have beenclosed under vacuum in a low humidity environment after lyophilizationreconstitute especially quickly.

Microparticles which include ligands according to the invention areuseful in a wide variety of assays known in the art which utilizespecific binding between a ligand and its receptor for detection of ananalyte. Such assays include immunoassays and nucleic acid hybridizationassays which can be performed in several different formats, as is knownin the art. Certain of these assays include a tracer which becomesassociated with the complex formed by binding of the ligand to itsreceptor and thereby facilitates detection of the complex. Detection ofthe complex is an indication of the presence, absence or quantity of theanalyte, depending on the assay format. Conventional tracers comprise aligand (an antibody, antigen or oligonucleotide) coupled to a detectablelabel such as a dye, a fluorescent compound, a radioisotope or an enzymewhich can be reacted with a substrate to produce a colored product. Thetracers of the invention, in contrast, comprise functionalizedmicroparticles having the ligand associated with the surface such thatit is capable of binding to a receptor, and a dye included in thehydrophobic liquid core. While the tracer microspheres may besubstituted for conventional tracers in essentially any of the knownimmunoassay or nucleic acid hybridization assay formats, solid phaseimmunoassays are preferred because separation of reagents is faster andsimpler than in other assay formats. A preferred solid phase assaydevice is an immunocapillary or immunochromatographic assay device, asthe intensity of the dye color, the reduced background and fewer falsepositives associated with the microspherical tracers provide particularadvantages in these types of assays.

Immunocapillary or immunochromatographic immunoassay devices comprise amicroporous absorbent material such as nitrocellulose, nylon, methylacetate, methyl cellulose or glass fiber. Alternatively porous materialswhich are not substantially absorbant, such as certain ceramics, may beused in such devices employing a vaccuum to draw reagents and sampleinto the material. To perform an immunoassay using such a device, aportion of the microporous absorbent material is contacted with a fluidsample such that the sample is drawn up into the device by capillarity(wicking), thus bringing the sample into contact with a capture reagentimmobilized on the microporous absorbent at a position removed from thepoint of contact with the sample. The immobilized capture reagent isgenerally an antibody or antigen which serves to capture an antigen orantibody analyte in the sample by binding to it. In a competitive assayformat, the ligand and the analyte are generally the same molecule oranalogues which bind the same receptor. For a competitive assay thetracer and the sample are wicked up into the microporous absorbentsimultaneously and compete for binding to the capture reagent. In asandwich assay format, the analyte is generally a receptor for theligand. The analyte is captured first and the tracer is wicked up intothe microporous absorbent into contact with the captured analyte,binding to it as an indication of the presence of the analyte.

In one embodiment of an immunocapillary assay device employing thefunctionalized microparticle tracers of the invention, anti-malariaantibodies are coupled to the microparticles for detection of malariaantigen (the analyte) in a sandwich assay. An exemplary assay device isan immunocapillary dipstick having an anti-malaria capture antibodyimmobilized above the base of the stick. The end of the dipstick belowthe line of capture antibody is brought into contact with the sample tobe tested and the sample fluid is drawn by capillarity into contact withthe capture antibody. After binding of any analyte which may be presentin the sample to the capture antibody, a composition comprising themicroparticle tracer is drawn into contact with the captured malariaantigen, if present. Development of a detectable, preferably visible,line of dye in the region of the capture antibody is an indication ofthe presence of malaria antigen in the sample.

In a second embodiment, the functionalized microspherical particles(with or without incorporated dye) may be used in serologicalagglutination assays similar to the VDRL test for syphilis. In theseassays, an antigen associated with disease may be covalently coupled tothe surface of the microparticles or included in the amphiphilicmonolayer coating on the surface. Mixing the functionalizedmicroparticles with the serum of a patient which contains antibodies tothe disease associated antigen results in cross-linking of themicroparticles by antibody and visible particle aggregation.Alternatively, antibody may be coupled to the microparticles and used todetect an antigen present in serum by agglutination of themicroparticles.

The following experimental examples are presented to illustrate certainspecific embodiments of the invention but are not to be construed aslimiting the invention as defined by the appended claims.

EXAMPLE 1 Preparation of Silicone Microemulsions

A mixture of 47 mg of distearoyl phosphatidyl choline (DSPC, AvantiPolar Lipids, Pelham, Ala.), 10.3 mg. distearoyl phosphatidyl glycerol(DSPG, Avanti Polar Lipids), 1.87 mg distearoyl phosphatidylethanolamine-maleimidyl caproate (DSPE-MC), 59 mg Fast Sudan Black B(Aldrich Chemical Co., Milwaukee, Wis. or Eastman Kodak, Rochester,N.Y.), 236 μl polydiphenyldimethyl siloxane (GE Silicone SF 1154,General Electric Co., Waterford, N.Y.) and 8.87 ml 200 proof ethanol(dried with a 4A molecular sieve and filtered with a Millipore GVsyringe filter--Quantum Chemical Corp., USI Division, Tuscola, Ill.) washeated at 55° C. for 1 hour. The ethanol solution (1 part) was thenmixed with water (3 parts) using a Zymark Corp. master laboratorystation with 10 and 25 ml syringes controlled by stepper motors. Thefluid streams were mixed in a "Y" connection, resulting in instantaneousparticle formation. After formation of the microemulsions, ethanol wasremoved by dialysis against 10 mM EDTA and the microemulsions wereconcentrated by dialysis against a dessicant. The size of the particles,as determined by analysis with a Coulter Corp. (Hialeah, Fla.) model N4MD sub-micron particle analyzer, was about 187 nm by unimodal analysis.The phosphorous concentration was determined to be 3.4 pM Pi/ml, and themaleimide was determined to be 98.8 nMMC/ml.

EXAMPLE 2 Coupling of Microparticles to Antibody

The microparticles prepared in Example 1 were coupled to rabbitanti-recombinant HRP II antibody. These antibodies are directed againstthe histidine rich protein II antigen of Plasmodium falciparum, theetiological agent of malaria. Anti-HRP II antibody was dialyzed intophosphate buffered saline (PBS), pH 8, resulting in a concentration of2.47 mg/ml as determined by absorbance at 280 nm with an extinctioncoefficient of 1.35. Six mg of antibody was derivatized with SPDP(3-(2-pyridyldithio propionic acid N-hydroxysuccinimide ester, SigmaChemical Co., St. Louis, Mo.) for 30 min. using a solution of 1.5 mgSPDP in 1.5 ml methanol (60 μl). 1M sodium acetate, pH 4.5 (280 μl) wasthen added and followed by 86 μl of 1M dithiothreitol (DTT), resultingin a final DTT concentration of 33 mM. The solution was stirred at roomtemperature for an additional 30 min., then applied to a SEPHADEX G-25column (Pharmacia LKB Biotechnology, Inc.) to remove free DTT and placethe derivatized antibody into pH 8 coupling buffer (50 mM Tris Base, 50mM sodium acetate, 50 mM sodium chloride and 1 mMEDTA).

The concentration of antibody was determined absorbance at 280 nm. Theantibody preparation was mixed with the microemulsion, which had beenbrought to pH 8 with Tris immediately before use, at a ratio of 1 mgantibody per 100 nM maleimide. The mixture was incubated for 2 hours.The functionalized particles were then separated from free antibody on aSEPHAROSE FAST FLOW column (Pharmacia LKB Biotechnology, Inc.)equilibrated in 30 mM MOPSO, 10 mM EDTA, 100 mM glucose, and 0.2% sodiumazide, pH 6.8. Miles Pentex Fraction V BSA was added to give a final w/vconcentration of 1%.

EXAMPLE 3 Immunoassay Using Tracer Microspheres

Immunocapillary assay devices for detecting malaria antigen wereprepared as follows. A 2.8 cm×15 cm strip of 8 micron nitrocellulose(Schleicher and Schuell, Keene, N.H.) was laminated onto plastic. Amonoclonal capture antibody raised in response to an 18-mer peptide withan amino acid sequence derived from the tandem repeat sequence of HRP IIwas spotted onto the nitrocellulose using a 1 mg/ml solution of theantibody applied at a rate of 1 μg/cm². Two hybridomas which produce theanti-18mer antibody have been deposited with the American Type CultureCollection (Rockville, Md.) under Accession Numbers HB 11111 and HB11112. The membrane strips were then incubated at 45° C. for 15 min. andblocked with a solution of 5 mM EDTA, 5% beta lactose, 0.2% NaN₃, 0.2%ZWITTERGEN 3-10 (Calbiochem, LaJolla, Calif.) and 1% BLOTTO (nonfat drymilk). The blocked strips were placed at 45° C. overnight. The preparedmembranes were applied to a 7 cm×15 cm strip of laminated plastic andoverlapped with a 4.7 cm×15 cm piece of glass fiber (Gelman Sciences,Ann Arbor, Mich.) to assist in drawing fluids into the nitrocellulose.The strips were cut into 7 mm wide test sticks and stored dessicated.

For the assay, the malaria test sticks were placed in a test tube with40 μl of normal human serum (NHS) containing various dilutions of the18-met peptide as the analyte. The 18-mer is a unique synthetic peptidecomprising the following amino acid sequence, SEQ ID NO:1:

    Cys-Gly-Ala-His-His-Ala-His-His-Ala-Ala-Asp-Ala-His-His-Ala-Ala-Asp-Ala.

The microparticle tracer prepared in Examples 1 and 2 was diluted 1:3with MOPSO buffer containing 1% BSA and 40 μl of the diluted tracer wasadded to the tube after the serum sample had been wicked up into thetest stick. After allowing the tracer to be drawn into the test stick,40 μl of a wash solution (pH 7.5 borate buffer with 0.2% ZWITTERGEN3-10) was added and also allowed to wick up. The degree of color in thearea of the capture antibody line was visually determined and assigned asubjective number based on the intensity. Endpoints for detection of thesignal were typically at antigen dilutions of about 1×10⁻⁷, indicating ahighly sensitive assay.

The test results are summarized in the following Table.

Preparation A (the microparticle tracer) is compared to a liposometracer encapsulating sulforhodamine B (Lot 002).

    ______________________________________                                        PARTICLE        DILUTION                                                      PREP.     NHS       10.sup.-4                                                                            10.sup.-5                                                                            10.sup.-6                                                                          10.sup.-7                              ______________________________________                                        A         --        4      3      2     1+                                    Lot 002   WK + 1    4      3      2    1                                      ______________________________________                                    

These results demonstrate that the assay using the microparticle tracersdetects a clinically relevant amount of malaria antigen and iscomparable in performance to a liposome tracer encapsulating dye.

EXAMPLE 4 Lyophilization and Reconstitution of Microparticles

Microemulsions prepared as in Examples 1 and 2 were stored in MOPSObuffer with 1% Miles Pentex Fraction V BSA. Aliquots of 166 μl wereplaced in vials with 333 μl of MOPSO buffer/1% BSA. Stoppers wereloosely placed on the vials. The vials were frozen in liquid nitrogenand placed on a frozen shelf in a lyophilizer (The Virtis Co., Inc.,Gardiner, N.Y.). The lyophilization cycle was controlled such that theshelf warmed from -40° C. to +25° C. over a 12 hour period under vacuum.The condensor remained at -40° C. The vials were held at 25° C. untilthe run was complete, a minimum of approximately 18 hours. The vialswere then stoppered in the lyophilizer, under vacuum and at lowhumidity, by filling a bladder under the tray with room air. Thestoppered vials were stored for 11 days at 45° C.

To reconstitute the microemulsions, the stopper was removed and a volumeof MOPSO buffer/1% BSA was added to correspond to the desired dilutionof particles. For use in the immunoassay of Example 3, 500 μl of bufferwas added to reconstitute the microemulsions. Rehydration occurredquickly, forming microemulsions containing 238 nm particles. Activity ofthe reconstituted microemulsion tracers was determined in an immunoassayas in Example 3. The results were as follows:

    ______________________________________                                        NHS        1 × 10.sup.-4                                                                    1 × 10.sup.-5                                                                     1 × 10.sup.-6                                                                  1 × 10.sup.-7                      ______________________________________                                        ACTIVITY                                                                              +/--   +4       +2      +1     +/-                                    ______________________________________                                    

EXAMPLE 5 Cardiolipin Microspheres

Phosphatidyl choline (10 mg, either DSPG, Avanti Polar Lipids or L-alphamixed chains, Sigma Chemical Co.) was mixed with 1 mg DSPG, 40 μl SF1154 silicone, 50 μl cardiolipin (Sigma Chemical Co., 5.2 mg/ml inethanol) and 1.5 ml ethanol. DSPG is optional. The solution was heatedat 55° C. for 1 hour, then added dropwise through a Gelman 4450 0.2 μm13 mm filter to 4.5 ml water mixing on a vortex. The resultingmicroemulsions contained 174 nm particles. The microemulsions weredialyzed in SPECTRA/POR 2 dialysis bags (Spectrum Medical Industries,Inc., Los Angeles, Calif., 12-14000 MW cutoff) against 10 mM EDTAovernight to remove ethanol.

Commercially available VDRL antigen (Difco Laboratories, Detroit, Mich.)contains 0.03% cardiolipin, 0.21% lecithin and 0.9% cholesterol inethanol. This ethanol solution was added to formaldehyde bufferedsaline, forming a colloidal suspension. The suspension was centrifugedand resuspended in 0.02M phsophate buffer, pH 6.9 containing 0.2%merthiolate, 40% choline chloride and 0.1M EDTA. This preparation wasreferred to as "USR reagent." Both the USR reagents and themicroemulsion were used to test sera for reactivity against cardiolipinas an indicator of exposure to syphilis.

One drop of the microemulsion or USR reagent delivered through an 18gauge needle onto a ringed glass slide containing 50 μl of saline, serumor serum diluted in saline. The slide was rotated at 180 RPM for 4 min.,and agglutination was read microscopically at 100× magnification. Thehighest dilution showing visible agglutination was determined to be thetiter. Representative data is shown in the following table:

    ______________________________________                                                     Silicone                                                                      Microemulsions                                                                             USR                                                 ______________________________________                                        Antiserum 110  neg            neg                                             Antiserum 114  neg            pos 1:2                                         Antiserum 115  pos 1:2        pos 1:2                                         Antiserum 119  pos 1:8        pos 1:8                                         ______________________________________                                    

These results show that, using antiserum of known reactivity, in mostcases the cardiolipin microparticle preparations exhibit reactivitysimilar to the standard, commercially available syphilis serology test.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CysGlyAlaHisHisAlaHisHisAlaAlaAspAlaHisHisAlaAla                              151015                                                                        AspAla                                                                        __________________________________________________________________________

What is claimed is:
 1. A method of making functionalized microsphericalparticles comprising:a) preparing a solvent solution comprising a watermiscible organic solvent, a liquid silicone compound and an amphiphilicligand, and; b) combining said solvent solution with an aqueous mediumto form an oil-in-water microemulsion having as the dispersed phasemicrospherical particles comprising a liquid silicone core and amonolayer on the surface of the core comprising the amphiphilic ligand.2. The method of claim 1 wherein said solvent solution further comprisesa water-insoluble dye.
 3. The method of claim 2 wherein said solventsolution further comprises a phospholipid selected from the groupconsisting of phosphatidyl choline, phosphatidyl glycerol, phosphatidylethanolamine and distearoyl derivatives thereof and said liquid siliconecompound is selected from the group consisting of polydimethyldiphenylsiloxanes and fluorosilicones.
 4. The method of claim 2 wherein saidamphiphilic ligand is a cardiolipin.
 5. The method of claim 1 furthercomprising removing the water miscible organic solvent after combiningthe solvent solution with the aqueous medium.
 6. The method of claim 1wherein said solvent solution and the aqueous medium are combined at aY-shaped connector using automated means for delivering the solventsolution and the aqueous medium at a preselected ratio of solventsolution to aqueous medium.
 7. The method of claim 6 wherein theautomated means comprises motor-controlled syringes or pumps havingseparately controllable speeds.
 8. The method of claim 1 wherein thewater miscible organic solvent is an alcohol.
 9. The method of claim 1further comprising lyophilizing the microspherical particles.
 10. Amethod of making microspherical particles comprising:a) preparing asolvent solution comprising a water miscible organic solvent, a liquidsilicone compound and an amphiphilic compound, and; b) combining saidsolvent solution with an aqueous medium to form an oil-in-watermicroemulsion having as the dispersed phase microspherical particlescomprising a core, the core comprising the liquid silicone compound, anda monolayer on the surface of the core comprising the amphiphiliccompound, and; c) isolating the microspherical particles.
 11. The methodof claim 10 wherein said solvent solution further comprises awater-insoluble dye.
 12. The method of claim 11 wherein said liquidsilicone compound is selected from the group consisting ofpolydimethyldiphenyl siloxanes and fluorosilicones, and said amphiphiliccompound is a phospholoipid selected from the group consisting ofphosphatidyl choline, phosphatidyl glycerol, phosphatidyl ethanolamineand distearoyl derivatives thereof.
 13. The method of claim 12 whereinthe water miscible organic solvent is an alcohol.
 14. The method ofclaim 10 further comprising lyophilizing the microspherical particles.15. The method of claim 10 wherein the solvent solution and the aqueousmedium are combined at a Y-shaped connector using automated means fordelivering the solvent and the aqueous medium at a preselected ratio ofsolvent solution to aqueous medium.